Coronaviruses are a family of enveloped RNA viruses that cause respiratory, enteric, and neurologic diseases in mammalian and avian hosts. In humans, four coronaviruses are responsible for common upper respiratory tract infections; a fifth human coronavirus is the causative agent of severe acute respiratory syndrome (SARS), which has the potential to re-emerge into the population from animal reservoirs. The overall goal of this proposal is to delineate mechanisms that are critical to multiple phases of coronavirus replication, using the prototype coronavirus mouse hepatitis virus (MHV). Coronaviruses have the largest genomes of all RNA viruses and their basic molecular biology is consequently intricate. There thus remain numerous gaps in our knowledge of the essential events at the earliest stages of infection, following entry of the viral nucleocapsid into the host cell, and at late stages of infection, when progeny envelope proteins and nucleocapids combine for budding. To manipulate the genomes of coronaviruses, we developed the earliest reverse genetic system, via targeted RNA recombination. This robust and versatile technique has been an indispensable tool for analyses of MHV structural proteins. More recently, full-length coronavirus cDNA systems have also become available, allowing access to the viral replicase. These powerful reverse genetic methodologies, together with complementary biochemical and molecular biological approaches, will be employed to accomplish three specific aims. (1) We will investigate a newly discovered critical interaction between the nucleocapsid (N) protein and a component of the viral replication-transcription complex (nsp3). This will entail identification of the particular modules of the huge nsp3 molecule that are essential for viral replication. (2) We will elucidate the crucial role of N protein in the initiation of coronavirus infection, which, we hypothesize, is to deliver the viral genome to the nascent replication-transcription complex. Moreover, we will learn how this role is modulated by phosphorylation of N protein. (3) We will further dissect the network of structural protein interactions among N protein, the membrane (M) protein, and the small envelope (E) protein that are essential to virion assembly. These efforts will build upon our discovery of unusual M protein variants that evolve in E deletion mutants, and will use interspecies M protein chimeras to probe M-M and M-N interactions. Additionally, we will explore the role of the genomic packaging signal in the selective incorporation of the nucleocapsid into virions.